Based on a progress of a technological development regarding a recent protein translation system, a synthesis of various peptides and proteins incorporating non-natural amino acids is capable. For developing a new bioactive substance and creating a technological high-performance nano-device, these non-natural compositions become an important technical base. However, a shortage of non-natural basic unit applicable to the translation system in a cell has been an obstacle for synthesizing a wide variety of non-natural peptides and non-natural proteins.
For adding new function to a protein, it is pointed out that not only a side chain structure of a polypeptide but also a structure of the polypeptide backbone play an important role. For example, it is known that once one amide bond is replaced by an ester bond at a tranmembrane domain of membrane protein, a hydrogen bond derived from a peptide bond of the backbone is destroyed, which affects great influence on permeability of the protein into the membrane and agonist activity (see, for example, non-patent document 1). There are several methods to replace the amide bond of the polypeptide backbone into the ester bond site-specifically in order to synthesize such non-natural proteins.
A first method is to introduce the α-hydroxy acid into a polypeptide based on the genetic code, in vitro or micro-injectionable cell (see, for example, non-patent documents 2 and 3). The α-hydroxy acid is introduced into in vitro translational system by binding it to tRNA corresponding to non-sense codon (an amber codon, a 4 characters codon, and the like) according to not enzymatic but organic chemical reaction. Thereby, the α-hydroxy acid is introduced into the polypeptide with the translational reaction in the ribosome at non-sense codon specifically. However, in this method, once a tRNA bound with the α-hydroxy acid is used in the reaction, the tRNA itself needs not to be reused. Accordingly, tRNAs (20-40 μM) bound with the α-hydroxy acid more than the number of the molecules of the polypeptide to synthesize must be synthesized with organic chemical reaction, and introduced into a test tube. Its organic chemical manipulation is so complicated and takes a cost. In addition, a polypeptide yield including the α-hydroxy acid synthesized in such system is very low.
Secondly, there is a method of in vitro introduction of the α-hydroxy acid into a polypeptide based on a genetic code using a ribozyme (see, for example, non-patent document 4). The α-hydroxy acid is introduced into in vitro translational system by binding it to tRNA corresponding to non-sense codon (an amber codon, a 4 characters codon, and the like) according to enzymatic reaction. Thereby, the α-hydroxy acid is introduced into the polypeptide with the translational reaction in the ribosome at non-sense codon specifically. However, in this method too, once the tRNA bound with the α-hydroxy acid is used in the reaction, the tRNA needs not to be reused. Accordingly, there is the same problem as described above, concerning the manipulation complicatedness and the production yield.
Thirdly, an introduction technique of non-natural amino acids within a living cell using the aminoacyl-tRNA synthetase (aaRS) and the tRNA is raised. In this method, an extraneous aaRS originally recognizing non-natural amino acid or artificially modified to recognize non-natural amino acid and a gene of a suppressor tRNA which is recognized by the above extraneous aaRS and recognizes a non-sense codon, are introduced into a living cell like E. coli and expressed in the cell. Thereby, the non-natural amino acid is enzymatically bound to the suppressor tRNA within the cell, and the non-natural amino acid is site-specifically introduced into a peptide (a protein) by the translational reaction in the ribosome. As a combination of the aaRS and the tRNA, an achaebacterium tyrosyl-tRNA synthetase (TyrRS) and an amber suppressor tRNATyr mutant, a lysyl-tRNA synthetase and a tRNA corresponding to 4 characters codon, a pyrrolysyl-tRNA synthetase (PylRS) and a tRNAPyl (see, for example, patent document 1) are used in E. coli, and a bacterium TyrRS and an amber suppressor tRNATyr mutant (see, for example, non-patent document 5), a leucyl-tRNA synthetase and an amber suppressor tRNALeu mutant, the PylRS and the tRNAPyl are used in eukaryote. However, in either enzyme, because of a specificity against an α-amino acid inherited in the aaRS, it has not succeeded yet in introducing the α-hydroxy acid so far, even if it has succeeded in introducing the α-amino acid having different side chain with artificial modification. In addition, it has been considered that such modification is actually difficult.
Fourthly, it is reported as an example that the α-hydroxy acid is introduced into a protein with a special in vitro translational system, in which the α-hydroxy acid corresponding to methionine is bound to the corresponding tRNA enzymatically using E-coli methionyl-tRNA synthetase (see, for example, non-patent document 6). This method is efficient because the α-hydroxy acid is bound to tRNA enzymatically many times in the reaction solution of in vitro translational system via a cycle that tRNA is used in ribosome. However, there are problems that the translational system itself should have a special composition of the aminoacyl-tRNA synthetase and the tRNA, and the α-hydroxy acid is introduced into all methionine codons. Moreover, this can not be used in a living cell.    [Patent Document 1] Japanese Patent Kokai Publication No. JP2007-037445A    [Non-Patent Document 1] England P M, Zhang Y, Dougherty D A, Lester H A. “Backbone mutations in transmembrane domains of a ligand-gated ion channel: implications for the mechanism of gating”, Cell 96, 89-98 (1999)    [Non-Patent Document 2] Koh J T, Cornish V W, Schultz P G. “An experimental approach to evaluating the role of backbone interactions in proteins using unnatural amino acid mutagenesis”, Biochemistry 36, 11314-11322 (1997)    [Non-Patent Document 3] Chapman E, Thorson J S, Schultz P G. “Mutational analysis of backbone hydrogen bonds in staphylococcal nuclease”, J. Am. Chem. Soc. 119, 7151-7152 (1997)    [Non-Patent Document 4] Murakami H, Ohta A, Ashigai H, Suga H. “A highly flexible tRNA acylation method for non-natural polypeptide synthesis”, Nat. Methods 3, 357-359 (2006)    [Non-Patent Document 5] Wang L, Brock A, Herberich B, Schultz P G. “Expanding the genetic code of Escherichia coli”, Science 292, 498-500 (2001)    [Non-Patent Document 6] Hartman M C, Josephson K, Lin C W, Szostak J W. “An expanded set of amino Acid analogs for the ribosomal translation of unnatural peptides”, PLoS ONE 2, e972 (2007)
The entire disclosures of the above patent document 1 and non-patent documents 1-6 are incorporated herein by reference thereto. The following analysis is given by the present invention.
As described in the above, the basic unit which can be linked has been only amino acid or derivative thereof in the in vivo translational system from the point of view in a substrate specificity of the aaRS. However, if the peptide bond of the backbone composing protein is replaced with the ester bond efficiently using in vivo translational system in the ribosome, it will be useful for synthesizing a variety of non-natural proteins.
In a process of researching the structure and functions of the aaRS, inventors of the present invention have found that the pyrrolysyl-tRNA synthetase (PylRS) which is an aminoacyl-tRNA synthetase inherited in partial achaebacterum and bacterium recognizes the α-hydroxy acid within E. coli and mammal cells, and the α-hydroxy acid could be specifically introduced into a stop codon of mRNA. And, using such a reaction system, they succeeded in enzymatic synthesizing a non-natural protein including an ester bond in a desired position in a polypeptide backbone within a living cell for the first time.
That is to say, a method of producing an unnatural protein of the present invention is characterized in that: expressing
(a) an aminoacyl-tRNA synthetase capable of activating an α-hydroxy acid,
(b) a suppressor tRNA capable of binding to the α-hydroxy acid in the presence of said aminoacyl-tRNA synthetase, and
(c) a gene encoding a desired protein that has a nonsense mutation or frameshift mutation at a desired site,
in a cell or cell extract solution in the presence of said α-hydroxy acid.
It is preferred that the aminoacyl-tRNA synthetase is a pyrrolysyl-tRNA synthetase (PylRS) derived from archaebacterium, or a mutant thereof. Further, it is preferred that the mutant pyrrolysyl-tRNA synthetase comprises an amino acid substitution of at least one amino acid residue selected from alanine at position 302, tyrosine at position 306, leucine at position 309, asparagine at 346, cysteine at position 348, tyrosine at position 384 and tryptophan at 417, in the amino acid sequence of a wild type pyrrolysyl-tRNA synthetase represented by SEQ ID NO:2.
In a preferred mode of the present invention, the amino acid substitution in the mutant pyrrolysyl-tRNA synthetase comprises at least one amino acid substitutions of: from alanine to phenylalanine at position 302; from tyrosine to alanine at position 306; from leucine to alanine at position 309; from asparagine to serine at position 346; from cysteine to valine or isoleucine at position 348; and from tyrosine to phenylalanine at position 384. In one mode, the mutant enzyme defined in the present invention is PylRS (Y384F), PylRS (Y306A), PylRS (L309A, C348V), and PylRS (A302F, Y306A, N346S, C348I, Y384F).
In another preferred mode, the α-hydroxy acid is an N-substituted 6-amino-2-hydroxyhexanoic acid, or an optionally substituted 3-phenyllacetic acid. It is further preferred that the substituent at amino group of the N-substituted 6-amino-2-hydroxyhexanoic acid is t-butoxycarbonyl group, or an optionally substituted phenyl group, benzyl group or benzyloxycarbonyl group. In one mode, the α-hydroxy acid defined in the present invention is 6-amino-N-Boc-2-hydroxyhexanoic acid, 6-amino-benzyloxycarbonyl-2-hydroxyhexanoic acid, 3-phenyllacetic acid.
When the α-hydroxy acid is introduced into the polypeptide, it is bound as an ester bond which is different from the usual amide bond. Compared to the amide bond, the ester bond can be easily cleaved by acid or alkali. Thus, it is applicable to prepare (and the like) a cleavable tag depending on pH at a desired position in a protein. Moreover, it is possible to modify a conformation of the protein into a new conformation that can not be achieved in amide bond. Because the ester bond can be introduced based on genetic information, an ester bond-introduced polypeptide having a certain (or specific) activity can be selected by a genetic screening method. Among such polypeptides, it is considered that there is a polypeptide having a new beneficial function that a conventional polymer consisting of only amino acids does not have.